One of the most important knowledge gaps in HIV/SIV vaccine research relates to the biological properties of viruses that are responsible for mucosal transmission. Within this HIVRAD consortium, this project will address this priority area by taking advantage of our laboratory's recent discovery of a novel strategy for identifying mucosally transmitted HIV-1 and SIV viral genomes (Salazar et al., J Virol, 2008; Keele, PNAS, 2008;Salazar, J Exp Med, 2009;Keele, J Exp Med, 2009). This strategy, which is based on single genome amplification, sequencing and analysis of plasma viral RNA within the context of a model of random virus evolution, identifies those viruses that are actually responsible for transmission and productive infection. This innovation, in turn, makes possible for the first time the molecular cloning and biological characterization of such transmitted/founder virus genomes. In this project, we propose to extend this enabling technology to the SIV-macaque infection model to test the following hypothesis: Molecular clones of transmitted/founder SIVs will reveal the particular properties of cell tropism, receptor and co-receptor usage, and neutralization phenotype that are necessary and sufficient for successful transmission via intrarectal, intravaginal and intravenous infection routes. Specific aims include: 1. To identify and enumerate transmitted/founder viruses in Indian rhesus macaques following intrarectal, intravaginal and intravenous infection with new genetically diverse SIVsmm strains. To moleculariy clone full-length genomes of transmitted/founder viruses responsible for productive infection by intrarectal, intravaginal and intravenous routes. To determine the in vitro biological properties of molecular clone-derived transmitted/founder viruses. To prepare stocks of transmitted/founder viruses for in vivo testing in Indian rhesus macaques. Results from these studies will shed new light on the molecular mechanisms underiying mucosal HIV/SIV transmission, identify new targets for vaccine-elicited immune responses, and generate a much needed set of new genetically-diverse SIV challenge strains for transmission, pathogenesis and vaccine research.